The influence of Cadmium is investigated in several concentrations on the cell biomass and biosynthesis of tropan alkaloids in suspension and transformed root cultures of Datura stramonium L. It is established that the presence of low concentrations of cadmium does not cause a dramatic decrease of cell biomass, however increases the content of total alkaloids and decreases the composition of tropan alkaloids.

Toxic metal pollution of soils has become a major environmental problem. Datura stramonium can become an interesting crop for the extraction of toxic metals from polluted soils because even natural populations show a tolerance. Moreover they belong to the group of accumulators and are a source of tropan alkaloids with pharmaceutical value. In this study we simultaneously studied the plant tolerance against toxic metals and the by toxic metals induced changes in the synthesis of secondary metabolites in suspension cultures of Datura stramonium L., because of their relatively rapid propagation of biomass, the relatively homogenous cell types in the culture system and the ability to easily manipulate culture conditions.

Establishment of suspension cultures

The suspension cultures were obtained from undifferentiated calli cells. Callus cultures were initiated from young leaves and stems of sterile plant Datura stramonium L., using MS media modified with addition of kinetin 2.0 mg/l, 2,4-D 0.1 mg/l, IAA 0.2 mg/l, sucrose 3%, agar 0.9% (Media G48). The calli material was rubbed trough a netlike surface into the flask and 50 ml liquid G48 media was added. Suspension were shacked continually (80 ppm), at 25^oC in the dark and subcultured at 15 days before harvest. The biomass was lyophilized before extraction. They have been grown on liquid medium with increasing concentration of toxic metal.

The plant material and hairy roots were lyophilized and ground. They were extracted as described earlier, with 0.2M sulfuric acid for 2 hr. After centrifugation the residue was washed with 25 ml acids. The combined acid filtrates, made alkaline (pH 12) by addition of 1M NaOH were extracted with CHCl₃. The chloroform-extracts were taken to dryness under vacuum.

A similar procedure with the omission of the initial step (H₂SO₄) was used to extract the alkaloids from the medium.

The alkaloid content was analyzed from the dry root materials and media, and expressed as sum of the cells and medium.

HPLC analysis

The alkaloid extracts were analyzed using an HPLC method on C6 column. The solvent system used was acetonitrile/5% H₃PO₄. Flow rate was 1 ml/min. The alkaloids were detected by UV-detector at 210 nm. Standard scopolamine hydrochloride and hyoscyamine hydrobromide were used to calibrate the column.

GC-MS analysis

The identification of alkaloids was confirmed by GC-mass spectrometry with Dr. L.Witte (Pharmaceutische Biologie, Braunschweig, Germany) as described in earlier (3,4). The GC-MS spectra were compared to the value for authentic alkaloids.

The growth characteristics of Cd treated suspension cultures (5 for each concentration) were no similar to that of the control. The fresh weights of suspension cultures with Cd was lower than weight of the control (Tab.1 and Tab.2). Inhibition of growth by 10, 20, 30, 40, and 50 mmol CdCl₂ reached 92%, 53%, 33% and 14% respectively, after 15 days. Nearly full inhibition of biomass yield was observed at 50 mmol. The growth index (GI) of all the suspension cultures shows a decreasing tendency compared to GI of the control. Further investigations were conducted to examine the maximum admissible concentration (MAC). That is the concentration at which suspension cultures continue to develop. For the suspension cultures of Datura stramonium MAC of 46 mmol was established (Tab.3) and Cd-resistant cell lines was selected by a stepwise increase in Cd concentration. They are capable of growth and division in normally lethal concentration of Cd. Cells from the resistant cultures were converted to protoplasts by enzymatic digestion and individual protoplasts were utilized to initiate new suspension cultures.

Thus eliminating the chance that a suspension culture was derived from a chimera, composed partially of resistant and partially of sensitive cells. The content of total alkaloids in suspension cultures (control) was 0.83 % at day 15. The alkaloid production in medium with CdCl₂ varied from 0.50 to 0.92 % (HPLC).

Treatment of suspension cultures of D. stramonium with Cd²⁺ ions in low concentration induced synthesis of tropan alkaloids. The best induction was achieved at about 20 mmol. While production was rapid over the first 9 days in medium with 44 mmol Cd, little or no further increment was found thereafter. We have demonstrated that, elicitation is however effective only by the low concentration of Cd. It has been establish in Datura stramonium cultures that elicitation of tropan alkaloids includes decrease of their composition (Tab.4 and Tab.5) and it seems probable that the seem mechanisms are active in D. stramonium hairy roots (1,2).

1. Ionkova I., Demeyer K., Witte L. and Alfermann A.W. (1998): Effect of Cadmium on the Formation of TropanAlkaloidsin HairyRoot CulturesofDatura stramonium L.,46. Annual Congress of Society for Medicinal Plant Research, Vienna, 31 August - 4 September, Austria

2. Ionkova I., Demeyer K., Zvetanova V., Ninov S. and Alfermann A.W. (1999): Induction of Tropane Alkaloid Formation as a Result of Heavy Metal Toxicity: Dose-Response Relation in Suspension and Transformed Root Cultures of Datura stramonium L. Treated with Cadmium, Second Pharmaceutical Congress of Republic of Macedonia, Struga,Oktober, Macedonia

3. Ionkova I.,Witte L. and Alfermann A.W. (1994): Spectrum of Tropan Alkaloids in Transformed Roots of Datura innoxia and Hyoscyamus x gyoerffyi Cultivated in vitro, Planta Medica, 60 382-384.

4. Ionkova I. (1992): Alkaloid Production of Hyoscyamus reticulatus Plant and Transformed Root Culture Clone, Biotechnology & BioE (BG), vol. 6(2), 50-52.